Effect of different types of cryoprotectants on developmental capacity of vitrified-thawed immature buffalo oocytes

Studies were conducted to compare viability of immature buffalo oocytes vitrified in different types of cryoprotectants on the post-thaw morphological appearance, the in vitro maturation and developmental competence of buffalo oocytes. The vitrification solution (VS) consisted of Dulbecco’s phosphate buffered saline (DPBS) supplemented with 0.5 M sucrose, 0.4% bovine serum albumin (BSA) and different types of molar (M) concentrations of the cryoprotectants which were composed of either glycerol (G), ethylene glycol (EG) or dimethyl sulfoxide (DMSO) in order to determine the best type of vitrification cryoprotectants. The concentrations tested were 4 M, 7 M and 7M concentration of G, EG and DMSO, respectively. Cumulus oocyte complexes (COCs) were obtained from slaughterhouse ovaries. Oocytes and their cumulus cells were vitrified immediately after collection .The COCs were preequilibrated in 50% of the VS for 3-5 min, then kept in VS for 1 min and loaded in pre-sterilized 0.25 ml straws for 7-10 days of storage in liquid nitrogen. The straws were thawed in warm water at 37°C for 10 s. The COCs were equilibrated in 0.5 M sucrose in modified phosphate buffer (M-PBS) for 5 min and then washed in washing medium (TCM-199 plus 10% FCS). Oocytecumulus cells were evaluated for morphological damage. Morphologically normal COCs (Oocytecumulus cells) were cultured in vitro and evaluated for maturation. Oocytes were fertilized with frozen-thawed semen capacitated in Brackett and Oliphant (BO) medium containing heparin and caffeine and were evaluated for cleavage and embryonic development. The results revealed that the proportion of buffalo oocytes found to be morphologically normal was significantly (P < 0.05) higher in EG and DMSO than those obtained in G (85.0 and 83.33 vs. 65.0%, respectively). Among the damaged oocytes, cracking of zona pellucida was the most frequent abnormality observed. A significantly higher (P < 0.05) percentage of maturation derived from vitrified -thawed immature oocyte in EG than those obtained in G (47.05, vs. 30.76%., respectively). A significantly higher proportion of oocytes were cleaved in EG and DMSO compared to those obtained in G (28.57 and 25.71 vs. 10.0%; P < 0.05, respectively). A similar trend was observed in blastocyst stage produced in vitro. However, in vitro developmental competence was higher for vitrified-thawed fresh oocytes (control) than those obtained from all groups of cryoprotectants. In conclusion, the 7 M solution of EG or DMSO could be used for vitrification of immature buffalo oocytes for their subsequent utilization in the in vitro maturation, fertilization and embryo production.